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Phytochemical Constituents and in vitro Antioxidant Capacity of Methanolic Leaf

Extract of Oxytenanthera abyssinica (A.Rich Murno) Determined at Varying

Concentrations of The Plant Extract.

Ibeh Bartholomew O.1* and Ezeja Maxwell I.2

1. Department of Biochemistry, College of Natural and Applied Sciences, Michael Okpara University of

Agriculture P.M.B.7267 Umudike Abia State Nigeria. barthokeyibeh@yahoo.com.

2.Departmnent of Veterinary Physiology, Biochemistry and Pharmacology, College of Verterinary

Medicine, Michael Okpara University of Agriculture Umudike Abia State Nigeria.

ezejamaxwell@yahoo.com,

Authors’ Contribution to the Work

Dr Bartholomew O. Ibeh: Conceptualized and designed the work and interpretation of results.

Laboratory analysis and drafting of the original manuscripts and final approval of the version.

Dr Ezeja Maxwell: Involved in the project design, involved in result interpretation and laboratory

analysis. Critical revision of draft article for suitability and intellectual content and final approval of the

version.

Conflict of Interest: There is no potential conflict of interest.

Funding Received: None

*Guarantor/Correspondence: Dr. Bartholomew. O. Ibeh1.Correspondence and request for

materials should be addressed to Dr. B.O.Ibeh, P.O. Box 17919 Gariki, Abuja, FCT, Nigeria.

(E-mail:barthokeyibeh@yahoo.com Ph:+2348068767253

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ABSTRACT

Aim: The antioxidant and phytochemical properties of leaf extract of the Nigerian Oxytenanthera

abyssinica was evaluated at different concentrations using ascorbic acid standard. This preliminary

investigation into the bioactive component of the plant may reveal the biochemical and pharmacological

mechanism of action of the herb thus renew research into its’ medicinal rather than economic values.

Methodology: The antioxidant properties was determined using three assay models; the 2,2-diphenyl-1-

picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP) colorimetric test and a modified

version of in vitro antioxidant TBARS after cold extraction maceration. The activity was determined at

different concentrations (10µg/ml,50µg/ml,100µg/ml,200µg/ml and 400µg/ml) of the extract expressed

as vitamin C equivalent. Phytochemicals were determined by standard detection and spectrophotometric

methods.

Results:The yield of the methanolic leaf extract of Oxytenanthera abyssinica was 3.92% w/w dry. The

presence of steroids (steroid glycoside), alkaloids, saponins, tannins cardiac glycosides, flavonoids,

phlobatanins, anthroquinone and terpenes was detected while cyanogenic glycosides was absent. The

quantitative analysis yielded flavonoids (1.51±0.23), alkaloids (1.40±0.02),saponin(1.1±0.10),

polyphenols (1.31±0.32), tannins (0.071±0.40) and saponins (1.2±0.10). Oxytenanthera abyssinica’s

DPPH reduction was highest at 400µg/ml (82.10 + 0.01%) concentration with IC50 value of 56.2µg/ml.

The ferric reducing power of the extract at 400µg/ml was 61±1.52% (FRAP:0.61) and that of inhibition

of lipid peroxidation (measured as TBARS) was 81.±1.11%.

Conclusion: There is an indication that Oxytenanthera abyssinica have important phytochemicals with a

resultant antioxidant property comparable with standard antioxidant compounds that may link to its

therapeutic efficacy.

Keywords: Oxytenanthera abyssinica, Antioxidants, phytochemicals, flavonoid, medicinal plant,

Nigeria

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1.0 Introduction

Phytochemicals may be described as non-nutritive plant chemicals that have protective or disease

preventive properties. They are regarded as non essential nutrients (Okwu and Okwu, 2004). Normally,

they are naturally occurring bioactive molecules produced by plants to for protection from the elements

of the earth and the sun’s harmful rays. These amazing phyto-compounds provide food resources for the

body’s many cells. Consumption of plant foods by humans containing these compounds have been

scientifically validated to help slowdown the aging process and reduce the risk of many diseases

including; cancer, heart disease, stroke, high blood pressure, cataracts, osteoporosis, diabetes and urinary

tract infections (Liu, 2004; Rao and Rao, 2007; Barjesteh et al., 2007).

It has been noted that one of mechanism of action of phytochemicals is through antioxidative activity.

Oxidation, a chemical reaction that transfers electrons from a substance to an oxidizing agent produces

free radicals (Muller et al., 2007) and also initiates chain reaction that may damage cells (Marnett,

1999; Metcalfe and Alonso-Alvarez, 2010). Antioxidants terminate these chain reactions by removing

free radical intermediates and inhibit other oxidation reactions. They do this by being oxidized

themselves and are however, often reducing agents such as thiols, ascorbic acids and/or phenols (Stahl

and Sles, 1997). These antioxidants may be natural (ascorbic acid [AA, E300] and tocopherols [E306])

as well as synthetic (propyl gallate [PG, E310], tertiary butylhdroquinone [TBHQ], butylated

hydroxyanisole [BHA, E320] and butylated hydroxytoluene [BHT, E321] ) thus can be synthesize in the

body or obtained from the diet (Miller and Britigan, 1997 ; Vertuani, et al.,2004).

It is known that many herbs especially from tropical and subtropical climates have antioxidant activity

linked to their ethnomedicinal value. Oxytenanthera abyssinca is a tropical drought resistant plant

(bamboo) that grows in open grassland, lowlands and highlands, though mostly on hills or along

intermittent watercourses (Sharma, 1987; Burkill, 1994). They are distributed mainly in the tropics and

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also occur naturally in subtropical and temperate zones of all continents except Europe. The species are

found at latitudes 46° N to 47° S and from sea level to 4000m elevation (Ohrnberger, 1999). It is often

found on very poor soils in the sub-Sahara African (Kigomo and Kamiri,1985;1987) and can survive fire

in its natural habitat. Evidence indicates that each plant flowers once in its’ life time dies and does not

regrow spontaneously in the same region (Palgrave 1988; Inada & Hall, 2003 and 2008; Mgeni, 1983) .

In Nigeria, this herb is known ethnically by the following names; Nnyanyanga in Efik, Kewal in

Fulfulde, Kawu in Gwari, Goora in Hausa, Achala oyibo or Otosi in Igbo, Gamare in Kanuri, Eman in

Loke, Takarwa in Ngizim and Apako in Yoruba and Shuwa Arabic.

In sub-Sahara African countries, the bamboo has various uses such as roofing and furniture

construction, shelterbelts and windbreaks (whole plant), fuel for cooking and heating (charcool), split

stems for basket weaving, fresh/dried leaves for fodder and sap for wine making (Sath et al., 2008;

Asaolu et al., 2009; Mekuriaw et al., 2011 ). Also the stems are widely used for fencing and fish-traps,

stakes, drainages, trellises, tool handles, paper making, household implements, arrow shafts (Akinbile, et

al., 2011) and the seeds and young shoots as famine food (Antwi-Boasiako,et al., 2011). The bamboo of

this herb therefore is highly valuable to the local population. Current trend in parts of Africa shows that

the herb is used as a poverty eradication crop ( van de Ven and Van Cotthem, 2010) especially in sub-

Saharan African countries as contrasted by its’ high medicinal standing in the Chinese population.

Evidently, some African countries now fully protect the specie where harvesting is carefully controlled

and reintroduction programmes effectively established. In ethnomedicine, the rhizome is used in the

treatment of dysentery and the leaves marketed for treating diabetes, colic and rheumatism.

Oxytenanthera abyssinica has wide application in various countries for instance in Ethiopia the root is

applied in the treatment of skin diseases while in Senegal leaf decoctions are taken to treat polyuria,

oedema and albuminuria (Burkill, 1994; Louppe, et al., 2008).

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Generally, the economic potential of this herb in sub-Saharan Africa has masked its’ethnomedicinal

value hence current research efforts tend towards Oxytenanthera abyssinica’s potential as a poverty

alleviation plant. Therefore, we investigated for the first time the antioxidant and phytochemical

properties of the Nigerian Oxytenanthera abyssinica as a preliminary investigation in determining its’

bioactive compounds.

2.0 Materials and Methods

2.1 Collection and identification of plant materials

Leaves of Oxytenanthera abyssinca were plucked from the forest of Michael Okpara University of

Agriculture, Umudike, Nigeria. The leaves were identified and confirmed by experts of the department

of Forestry, College of Natural Resources and Environmental Management, Michael Okpara, University

of Agriculture Umudike, Nigeria. A voucher specimen with the number Ibeh 2010-59 was deposited in

the University herbarium for future reference.

2.2 Sample preparation

The leaves of Oxytenanthera abyssinca were first washed with distilled water to remove debris and

dust particles, air-dried at room temperature and pulverized into a uniform material using a Thomas-

Willey milling machine. Plant extraction was done by cold maceration of 300g in 80% methanol with

intermittent shaking at 2 hours interval for 48 hours. The extract was then filtered with Whatman filter

paper N0. 1 and the filtrate evaporated to dryness at 40ºC. The obtained crude extract was packed in

airtight plastic containers and stored in the in a refrigerator at 40C for the analysis.

The percentage yield of the extract was calculated using the formula:

% yield = ������ � ��� ���� �

������ � ����� ��������×

���

�

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2.3 Phytochemical determination

Phytochemical detection of major constituents of Oxytenanthera abyssinca was done using Thin-layer

Chromatography (TLC) on silica gel 60 F254 with layer thickness 0.25 mm(Dramstadt, Germany) after

dissolving the extract (2mg) in 2ml of the solvent. The plates were developed, then left to dry for about

10 minutes before they were viewed under UV fluorescence light at wavelength 254 and 366nm.

Spraying was done with the required detection reagent to determine the compounds present and the

solvent system which gave the best observation was presented in our results. For flavonoid, TLC was

developed in n-Butanol/acetic acid/water (4:1:5), then spots were visualized with 1% AlCl3 solution in

methanol under Ultraviolet (366nm) light. Alkaloids, saponins, tannins, anthraquinones, flavonoids,

terpenoids, steriods and cardiac glycosides were all identified based on the methods of Harbone,

(1973;1984) and Trease and Evans,(1987).Quantitative determination was carried out as previously

described by Trease and Evans,(1996), Sofowara,(2006) and Harborne (1998).

2.4 Antioxidant Assay

2.4.1 Inhibition of Lipid Peroxidation

A modified version of thiobarbituric acid reactive substances (TBARS) assay was used to determine

the level of lipid peroxide formed using egg yolk homogenate as lipid-rich media (Roberto and

Barrata,2000). Egg homogenate (0.5 ml, 10% v/v) was added to 0.1 ml of extract (1mg/ml) and the

volume made up to 1 ml with distilled water. Then 0.05 ml of FeSO4 was added and the mixture

incubated for 30 minutes. Acetic acid (1.5 ml) and thiobarbituric acid (1.5 ml) in SDS was sequentially

added. The resulting mixture was vortexed and heated at 95°C for 60 min. After cooling 5 ml of butan-

1-ol was added and the mixture centrifuged at 3000 rpm for 10 minutes. The absorbance of the organic

upper layer was measured at 532 nm and converted to percentage inhibition using the formula:

Inhibition of lipid peroxidation (%) = (1 – E/C) x 100

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Where C = absorbance of fully oxidized control and E = absorbance in the presence of extract

2.4.2 Ferric reducing antioxidant power (FRAP) assay

The reductive potential of Oxytenanthera abyssinca was determined according to the method of

Benzie and Strain,(1999) which is based on the chemical reduction of Fe3+ to Fe2+. At low pH, we

measured the reduction of ferric tripyridyl traizaine (Fe III TPTZ) complex to ferrous form by

monitoring the change in absorption at 593nm. The calculation was done by:

FRAP value of Sample (µM)=

(Change in absorbance of sample from 0 to 4 min) X FRAP value of standard (1000µM)

(Change in absorbance of standard from 0 to 4 min)

2.4.3 Determination of DPPH radical scavenging activity

Rapid thin layer chromatography (TLC) screening for antioxidant activity was carried out by spotting a

concentrated methanolic solution of the extract on silica gel plates. The plates were developed in

methanol: ethyl acetate (2:1) then air-dried and sprayed with 0.2% w/v DPPH spray in methanol. The

presence of yellow spot was detected. Radical scavenging activity of extracts was performed according

to the DPPH spectrophotometric method of Mensor et al.(2001) using Vitamin C (Emzor

Pharmaceutical Industries, Nigeria) as a reference antioxidant. Methanol (1.0 ml) plus extract solution

(2.5 ml) was used as blank while 1 ml of 0.3 mM DPPH plus methanol (2.5 ml) was used as a negative

control. The free radical scavenging properties of the extracts against 2,2-diphenyl-1-picryl hydrazyl

(DPPH) radical were measured at 518 nm, as an index to their antioxidant activity. The concentrations of

the extracts and Vitamin C used were 10, 50, 100, 200 and 400 µg mL-1 and the assay was carried out in

triplicates for each concentration. IC50 values ( the concentration of extracts required to scavenge 50%

of DPPH free radicals) were also obtained. The absorbance (Abs) of the resulting mixture measured at

518nm was converted to percentage antioxidant activity (AA %) and thus calculated by the equation:

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AA% = [100 – ((Abssample - Absblank) × 100)] /Abscontrol

2.5 Statistical analysis

The statistical analysis was done using one way analysis of variance (ANOVA). The differences

between the means were tested using Post Hoc LSD. A p-value of p< 0.05 was considered to be

statistically significant. We presented results as mean ± standard deviation

3.0 Results

3.1 Extraction yield

The yield of the methanolic leaf extract of Oxytenanthera abyssinica was 3.92% w/w dry matter.

3.2 Phytochemical analysis

Preliminary phytochemical screening of Oxyteneanthera abyssinica shows the presence of steroids

(steroid glycoside), alkaloids, saponins, tannins cardiac glycosides, flavonoids, phlobatanins,

anthraquinones and terpenes while cyanogenic glycosides was absent (table 1).The quantitative analysis

yielded the following phytochemicals; flavonoids (1.51±0.23), alkaloids (1.40±0.02) and polyphenols

(1.31±0.32) and moderate levels of tannins (0.07±0.40) and saponins (1.2±0.10 ) (table 2).

3.3 Antioxidant activity in vitro analysis

The percentage inhibition of DPPH by Oxytenanthera abyssinica and vitamin C, the ferric reducing

antioxidant power and the inhibition of lipid peroxidation measured as TBARS of the extract showed a

concentration-dependent antioxidant activity resulting from reduction of DPP (table 3), FRAP and

inhibition of TBARS (fig. 1) radicals to non-radical forms. Here the leaf extract of Oxytenanthera

abyssinica had a comparable DPPH reduction capacity at in all the extract concentrations measured

when compared with the scavenging activity of ascorbic acid. IC50 values for Oxytenanthera abyssinica

and ascorbic acid were 56.2 and 55.10 µg/ml respectively (table 3). The ferric reducing power of the

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extract at 400µg/ml was 61±1.52% (FRAP:0.61) and that of inhibition of lipid peroxidation (measured

as TBARS) was 81.±1.11% (fig. 1).

Table 1: Phytochemical screening of leaf extract of Oxytenanthera abyssinica

Plant Metabolite Leaf extract (L)

Cyanogenic glycosides -

Cardiac Glycoside ++

Steriod glycoside ++

Saponims +

Tanins ++

Alkaloid ++

Phlobatanins +

Terpenoids ++

Flavonoids ++

Anthroquinone +

L = Leaf extract of Axonopus compressus, + = Trace (insignificant amounts), +++ = Abundant, - =

Absent

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Table 2: Phytochemical composition of the leave extract of Oxytenanthera

abyssinica expressed as mg/100 g dry weight

Plant Metabolite Composition (mg/100g dry weight)

Polyphenols 1.31±0.32

Saponims 1.2±0.10

Tanins 0. 07±0.40

Alkaloid 1.40±0.02

Flavonoids 1.51±0.23

Results are mean of triplicate determinations on a dry weight basis ± standard deviation

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Table 3: Antioxidant activity measured as % reduction of DPP

Oxytenanthera abyssinica Ascorbic Acid

Concentration (µg/ml)

10 72.60 + 0.01* 73.64 + 1.82

50 73.85 + 0.01* 74.10 + 0.09

100 72.83 + 0.01* 74.62 + 3.46

200 79.97 + 0.01* 77.16 + 2.11

400 82.10 + 0.01* 87+ 0.11

56.2ǂ 55.1

ǂ

% Antioxidant Activity (Inhibition %)

*Showed no significant difference at (P<0.05)

ǂ IC50 value measured at µg/ml (concentration of sample required for

50% inhibition of DPPH radical activity).

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FIGURE 1 : FERRIC Reducing Potential (FRAP) and Inhibition of Lipid Peroxidation

(TBARS) by Oxytenanthera abyssinica

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4.0 DISCUSSION

For most regions in Africa Oxytenanthera abyssinica currently has major attraction as a natural product

amenable to sustainable management. It is grown throughout the tropical Africa outside the humid

forest zone. They are strong and the stems used as various construction materials, wine making, charcoal

for fuel energy and leaves for fodder (Embaye,2001). Nevertheless, despite wide rural significance

Oxytenanthera abyssinica remained neglected in the forestry and agricultural sectors until recently when

the International Network for Bamboo and Rattan (INBAR) included this specie among 38 priority

bamboos of economic importance (Rao, et al., 1998). The economic benefits of the plant have masked

its’ traditional ethno-medicinal exploits in the treatment of various ailments such as skin diseases, colic,

diabetes, rheumatism, poluria, oedema and albuminuria especially with the leaf decoctions. Scientific

investigations on the constituents of this plant with relevance to its ethno-medicinal value are scanty and

not well documented in Africa.

Our Thin-layer Chromatography (TLC) on silica gel 60 F254 revealed strong presence of phytochemicals

(tables 1 and 2). The concentration of phytochemicals obtained may indicate its ethno-medicinal use in

treatment of various diseases already noted. Our results are comparable with other known medicinal

plant phytochemicals such as nettle (Urtica dioica L.)(Khan et al., 2011) which have antioxidant and

antimicrobial effects (Gülçin et al., 2004).The therapeutic effect of this medicinal plant is evidently seen

on its use in the treatment of prostate hypertrophy, diabetes mellitus, rheumatic disease, hypertension,

osteoarthritis, diarrhea, rheumatoid arthritis, inflammation, skin disease and urinary tract infection

(Marrasinni et al., 2010). Generally, alkaloids are known to have antimicrobial, antifungal and anti-

inflammatory effects (Okwu and Okwu, 2004) and also acts as an anti-hypertensive agent (Sofowora,

1993). The folkloric use of Oxytenanthera abyssinica in the treatment of skin diseases, colic, diabetes,

rheumatism, poluria, oedema and albuminuria may owe its efficacy due to the presence and

concentration of these phytochemicals (tannins, alkaloid, saponins, glycosides, anthroquinones,

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terpenes, phlobatanins and polyphenols). These phyto-compounds have been linked to specific action

against infections and ailments (Sofowora, 1993).The absence of cyanogenic glycosides tend to support

its low or no toxicity content and considerable wide application as fodder and famine food sources.

Research has shown that at high experimental concentrations that are non-existent in vivo, the

antioxidant capabilities of flavonoids in vitro may be stronger than those of vitamins C and E though

depending on concentrations tested (Manashi et al., 1999). Phytochemical compounds mostly plant

phenolics are a major group of chemical species that act as primary antioxidants (Hatano et al., 1989).

They ultimately have high redox potentials which allow them act as reducing agents, hydrogen donors

and singlet oxygen quenchers (Kahkonen et al., 1999). This attribute is possible because of the

delocalization of electrons over the phenolics and stabilization by the resonance effect of the aromatic

nucleus that prevents the continuation of the free radical chain reaction as noted by Tsao and Akhtar,

(2005). From our results flavonoids and phenols were high in the methanolic leaf extract of the plant.

These are potent water soluble antioxidants which prevent oxidative cell damage suggesting their

possible implication in skin diseases, colic, diabetes and rheumatism treatment. Our results here

indicated a high and comparable antioxidant (inhibition of DPP [table 3] and TBARS [Fig 1] and FRAP

reduction reaction) capacity when we compared the scavenging strength of Oxytenanthera abyssinica

and ascorbic acid on DPPH radical. Plant phenolics therefore are major group of compounds acting as

primary antioxidants or free radical scavengers (Kahkonen et al., 1999). We used the DPPH assay

because its parameters are quick, reliable and reproducible to search for the in vitro general antioxidant

activity of pure compounds as well as plant extracts (Koleva et al., 2002). However, the decrease in

absorbance by the DPPH radical with concomitant increase in the concentration of the extract (table 3)

may have resulted in the rapid discolouration of the purple DPPH. This may suggest that the methanol

extract of Oxytenanthera abyssinica has antioxidant activity due to its proton donating ability. The data

presented here also showed that the antioxidant activity were concentration dependent having maximal

effect at 400µg/ml The DPPH activity obtained showed that our extract may have a comparable

15

antioxidant capacity with that of ascorbic acid requiring 56.2. µg/ml (IC50 value) to reach 50%

inhibition of DPPH radical activity though the value is higher than ascorbic acid (55.1 µg/ml). A lower

DPPH radical-scavenging activity is however, associated with a higher IC50 value. It has been shown

that the reduction mechanism of DPPH correlates with presence of hydroxyl groups on the antioxidant

molecules (Cotelle et al., 1996) which may suggest that the antioxidant activity of Oxytenanthera

abyssinica is probably due to the presence of substances with an available hydroxyl group such as

flavonoids or condensed tannins. All extracts at tested doses (100-400 µg mL-1) revealed good

scavenging activity for DPPH, FRAP and an appreciable inhibition of TBARS in a dose dependent

manner. Meir et al.(1995) noted that the reducing power of compounds could serve as indicator of

potential antioxidant properties. In the reducing power assay conducted, it is likely that the presence of

antioxidants in the extract reduced Fe3+ complex to the ferrous form with a reduction of about 61%.

This suggests that the leave extract is an electron donor and could neutralize free radicals.

Antioxidant is a widely consumed nutraceutical species common as food additives and also consciously

taken as a therapeutic agent in form of herbs. Antioxidants from natural resources such as extracts of

green tea, rosemary, oregano, liquorice and bamboo leaf is also being widely used in markets.

Phytochemical extracts exhibit strong antioxidant activity which may be from the combination of

phytochemicals present in the plant. It is of note that the numerous pharmacological actions of phenolic

compounds and flavonoids are linked to their ability to act as strong antioxidants and free radical

scavengers. As such the ethno-medicinal use of Oxytenanthera abyssinica may likely reside or be

attributed to the complex mixture of phytochemicals present. Furthermore, the therapeutic potential of

antioxidants in controlling degenerative diseases with marked oxidative damage from reactive oxygen

species or free radicals have been reported (Krishnamoorthy et al., 2005; Tripathi and Eka, 1999; Vinson

et al.,1995). These high antioxidant capacity and inhibition of lipid peroxidative activity may suggest its

potential in the treatment and prevention of various oxidative related diseases. Therefore, leave extract

of Oxytenanthera abyssinica could be exploited as sources of free radical scavengers and bioactive

16

metabolites for nutritional, medicinal and commercial purposes as Antwi-Boasiako1 et al. ( 2011)

recently reported its proximate composition.

5.CONCLUSION

Oxytenanthera abyssinica has demonstrated significant antioxidant activity in the model used and

showed strong presence of phytochemicals. The plant fundamentally possess bioactive ingredients and

bioactivity that may be responsible for its ethno-medicinal use thus should be exploited further.

Furthermore, detailed studies on the isolation and characterization of the plant phenols as well as in vivo

assays to discover novel biological antioxidants is needed.

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